Buoyant Density of DNA

Density gradient centrifugation

Density gradient centrifugation can be used to isolate DNA. The densities of DNAs are about the same as concentrated solutions of cesium chloride, CsCl (1.6 to 1.8 g/mL). Centrifugation of CsCl solutions at very high rotational speeds, where the centrifugal force becomes 105 times stronger than the force of gravity, causes the formation of a density gradient within the solution.

Isopycnic centrifugation.
This gradient is the result of a balance that is established between the sedimentation of the salt ions toward the bottom of the tube and their diffusion upward toward regions of lower concentration. If DNA is present in the centrifuged CsCl solution, it moves to a position of equilibrium in the gradient equivalent to its buoyant density. Caesium chloride is used because at a concentration of 1.6 to 1.8 g/mL it is similar to the density of DNA. For this reason, this technique is also called isopycnic centrifugation.

 The net movement of solute particles in an ultracentrifuge is the result of two processes: diffusion (from regions of higher concentration to regions of lower concentration) and sedimentation due to centrifugal force (in the direction away from the axis of rotation). In general, diffusion rates for molecules are inversely proportional to their molecular weightlarger molecules diffuse more slowly than smaller ones. On the other hand, sedimentation rates increase with increasing molecular weight. A macromolecular species that has reached its position of equilibrium in isopycnic centrifugation has formed a concentrated band of material.

 Cesium chloride centrifugation is an excellent means of removing RNA and proteins in the purification of DNA. The density of DNA is typically slightly greater than 1.7 g/cm3 (1.70+ 0.01), while the density of RNA is more than 1.8 g/cm3. Proteins have densities less than 1.3 g/cm3. In CsCl solutions of appropriate density, the DNA bands near the center of the tube, RNA pellets to the bottom, and the proteins float near the top.

 Single-stranded DNA is denser than doublehelical DNA. The irregular structure of randomly coiled ssDNA allows the atoms to pack together through vander Waals interactions. These interactions compact the molecule into a smaller volume than that occupied by a hydrogen-bonded double helix.

 Density of DNA is dependent on relative G : C content.

G : C-rich DNA has a significantly higher density than A :T-rich DNA.

Furthermore, a linear relationship exists between the buoyant densities of DNA from different sources and their G : C content

 The density of DNA, (in g/mL), as a function of its G : C content is given by the equation

where (GC) is the mole fraction of (G+ C) in the DNA.

For every 10% increase in GC content, the density rises by 0.12 units.

Effect of methylation
However, the presence of 5-methyl-cytosine serves to reduce the density slightly, thereby giving rise to an under-estimate of the GC content. In general, 1% methylation decreases the buoyant density by 1 mg cm3 .

 Furthermore, single-stranded DNA is denser than double-stranded DNA of similar base composition by approximately 0.015 g cm3 and under alkaline conditions the density is increased by 0.06 g cm3 . This is due partly to the fact that DNA becomes single-stranded under these conditions and also partly because the deprotonated adenine and thymine residues are neutralized by binding caesium ions.

Satellite DNA
Satellite DNA consists of highly repetitive DNA and is so called because repetitions of a short DNA sequence tend to produce a different frequency of the nucleotides and thus have a different density from bulk DNA - such that they form a second or 'satellite' band when genomic DNA is separated on a density gradient. For example, the fruit fly Drosophila virilis possesses a huge number of ACAAACT repeats that together add up to almost a quarter of the entire genome. The size of a satellite DNA ranges from 100 kb to over 1 Mb.

EtBr-CsCl centrifugation
ethidium bromide (EtBr) can be used to separate supercoiled DNA from nonsupercoiled molecules. Ethidium bromide binds to DNA molecules by intercalating between adjacent base pairs, causing partial unwinding of the double helix. This unwinding results in a decrease in the buoyant density, by as much as 0.125 g/cm3 for linear DNA.

 However, supercoiled DNA, with no free ends, has very little freedom to unwind, and can only bind a limited amount of EtBr. The decrease in buoyant density of a supercoiled molecule is therefore much less, only about 0.085 g/cm3. As a consequence, supercoiled molecules form a band in an EtBrCsCl gradient at a different position to linear and open-circular DNA